The long-term objective of this project is to define molecular mechanisms regulating exit from the mitotic cycle and entry into an alternative developmental pathway. Specifically, this proposal describes studies aimed at elucidating mechanisms used to regulate protein kinase activity. Protein kinases and their inhibitors are universally important elements of signal transduction pathways involved in growth and differentiation. Understanding protein kinase regulation is an approach to enable the development of novel chemical therapies designed to control cell growth. The aims of this proposal are to design structure/function studies of the Mei3 protein kinase inhibitor. Mei3 is a developmental activator in fission yeast. Expression of Mei3 is sufficient to divert cells from the mitotic cycle into the meiotic developmental pathway. Preliminary studies suggest that it belongs to the pseudosubstrate class of enzyme inhibitors. Thus, structure/function studies of Mei3 are expected to be applicable to a large number of known kinase inhibitory polypeptides. The specific aims of the project are to: (1) Characterize Mei3 inhibitory domain mutants. A series of eight alanine scanning mutants (SM mutants) have been constructed in the inhibitory region of Mei3. Two mutants fail to cause meiotic catastrophe. All mutants will be assayed for the ability to bind to and inhibit Ran1. (2) Investigate mechanisms used to regulate Mei3. Deletion analysis reveals that Mei3 is composed of two distinct domains. The carboxy-terminal half of the molecule is sufficient to cause meiotic catastrophe and to inhibit Ran-1 in vitro. The consequences of deleting the entire amino-terminal domain to normal meiosis will be examined in genetic studies. In parallel, the contribution of the amino-terminal domain to inhibition of Ran-1 in vitro. The consequences of deleting the entire amino-terminal domain to inhibition of Ran-1 will be investigated in biochemical experiments using recombinant proteins.